System For Additive Manufacturing Of Three-Dimensional Structures And Method For Same

ABSTRACT

A system and method for additive manufacturing of three-dimensional structures, including three-dimensional cellular structures, are provided. The system comprises at least one print head for receiving and dispensing materials, the materials comprising a sheath fluid and a hydrogel, the print head comprising an orifice for dispensing the materials, microfluidic channels for receiving and directing the materials, fluidic switches corresponding to one of the microfluidic channels in the print head and configured to allow or disallow fluid flow in the microfluidic channels; a receiving surface for receiving a first layer of the materials dispensed from the orifice; a positioning unit for positioning the orifice of the print head in three dimensional space; and a dispensing means for dispensing the materials from the orifice of the print head.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application claims priority under the Paris Convention from U.S.Application No. 61/834,420, filed on Jun. 13, 2013, the entire contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to three-dimensional (3D)printing and generation of three-dimensional biological structures fromdigital files. Specifically, the invention relates to a system,apparatus and method for fabricating 3D cell-laden hydrogel structures.

BACKGROUND OF THE INVENTION

3D printing, a form of additive manufacturing (AM), is a process forcreating three-dimensional objects directly from digital files. Softwareis used to slice a computer aided design (CAD) model or a 3D scan of anobject into a multitude of thin cross-sectional layers. This collectionof layers is sent to the AM system where the system builds thethree-dimensional object layer by layer. Each layer is deposited on topof the previous layer until the object has been fully constructed.Support material can be used to support overhanging and complex featuresof the object. Various AM processes exist that can build parts inplastic, metal, ceramic and/or biological materials.

Additive manufacturing could have applications in biological systems.For example, until recently, most cell culture studies were performed on2-dimensional (2D) surfaces, such as micro-well plates and Petri dishes.However, 2D culture systems do not mimic the 3D environment in whichcells exist in vivo. Researchers have found that 3D cell cultures behavemore like natural biological tissue than 2D cell cultures at least inpart because the 3D arrangement of cells in natural tissue influencescell-cell interactions, which in turn influences cell growth andphysiology.

Additive manufacturing devices and systems for fabricating cellularconstructs are known. For example, known fused fiber depositiontechniques have been applied to biological materials. In fused fiberdeposition, high viscosity liquids are dispensed from a relativelynarrow orifice and then rapidly solidified by a variety of means.Biocompatible plastics, thermal gelling hydrogels, UV-cross-linkablepolymers and high concentration alginates have been used as scaffoldsfor 3D cellular structures, wherein cells are added to the scaffoldafter it has solidified. A draw back to these techniques is that theyrequire cells to be added to the scaffold after printing, making itdifficult to control cell placement. Further, the composition of thescaffold substrates may not be appropriate for facilitating cellproliferation and growth.

Systems for printing 3D structures that comprise direct printing ofcellular materials are known and desired, at least in part, because theymay allow cells to be deposited within a 3D scaffold. For example, inkjet printing technology has been used to print biological materials.However, the shear force involved with propelling droplets of fluid ontoa substrate can damage cells dispersed in the fluid. Further, ink jetprinting is a slow process, which makes it challenging to adapt tobiological materials, which require specific environmental conditionsfor survival.

Other systems for directly printing cells within a 3D structure includeU.S. Pat. No: 8,639,484, which relates to use of a CAD model and a 3Dpositioning unit to deposit cellular materials through a multitude ofnozzles, layer by layer, to create a 3D object. Multiple nozzles allowfor multiple different materials to be included in the 3D object. USPatent Application Publication No: 2012/0089238 discloses a multicartridge print system for producing composite organic 3D structures,whereby the structure is built using at least two syringes, onecomprising a structural support polymer and another comprising a livingcell composition, that iteratively deposit the structural supportpolymer and living cell composition on a surface. US Patent ApplicationPublication No: 2014/0012407 discloses a device comprising one or moreprint heads, each configured to receive and hold one or more cartridges.Each cartridge comprises a fluid, such as a bio-ink comprising cells orsupport material, and an orifice wherefrom the fluid can be dispensedfrom the cartridge.

The prior art methods generally require requires multiple nozzles and/orcartridge orifices in order to facilitate printing of multiple differentmaterials (i.e., one material is dispensed by one nozzle or cartridgeorifice). Use of multiple nozzles for dispensing different materialsrequires a corresponding increase in movement of the printing system inorder to position the appropriate nozzle or cartridge orifice in acontrolled sequence to dispense a sequence of different materials. Suchincreased movement decreases speed and efficiency of printing.

It is desirable to obviate or mitigate one or more of the abovedeficiencies.

SUMMARY OF THE INVENTION

In a first aspect, a system for additive manufacturing ofthree-dimensional structures is provided. The system comprises at leastone a print head for receiving and dispensing materials, the materialscomprising a sheath fluid and a hydrogel. In one embodiment, the printhead comprises an orifice for dispensing the materials; microfluidicchannels comprising one or more first channels for receiving anddirecting the sheath fluid and one or more respective second channelsfor receiving and directing the hydrogel, the second channelsintersecting at a first intersection point with the first channels, thesecond and first channels joining together at the first intersectionpoint to form a dispensing channel which extends to the orifice; andfluidic switches, each fluidic switch corresponding to one of themicrofluidic channels in the print head and configured to allow ordisallow fluid flow in the microfluidic channels of the print head whenactuated. In one embodiment, the system further comprises a receivingsurface for receiving a first layer of the materials dispensed from theorifice; a positioning unit for positioning the orifice of the printhead in three dimensional space, the positioning unit operably coupledto the print head; and a dispensing means for dispensing the materialsfrom the orifice of the print head.

In one embodiment of the first aspect, the system comprises aprogrammable control processor for controlling the positioning unit andfor controlling dispensing of the materials from the print head onto thereceiving surface.

In one embodiment of the first aspect, the one or more first channelscomprise at least two channels, the one or more first channels beingconfigured to flank respective second channels at the first intersectionpoint.

In one embodiment of the first aspect, the sheath fluid comprises across-linking agent for solidifying the hydrogel upon contact therewithat the intersection point and/or in the dispensing channel.

In one embodiment of the first aspect, each second channel has adiameter less than that of the first channels and the dispensingchannel, whereby flow from the first channels forms a coaxial sheatharound the hydrogel in the dispensing channel.

In one embodiment of the first aspect, the hydrogel comprises livingcells.

In one embodiment of the first aspect, the system further comprises afluid removal feature for removing excess sheath fluid from dispensedfrom the print head.

In one embodiment of the first aspect, the receiving surface comprises aporous membrane comprising pores sized to permit passage of the excesssheath fluid there through.

In one embodiment of the first aspect, the fluid removal featurecomprises absorbent material or a vacuum for drawing the excess sheathfluid away from the receiving surface.

In one embodiment of the first aspect, the absorbent material or vacuumis applied below a porous membrane. In one embodiment of the firstaspect, the vacuum is applied above the receiving surface.

In one embodiment of the first aspect, the vacuum is applied through oneor more vacuum channels provided on the print head, the one or morevacuum channels having an orifice situated near the orifice of the printhead.

In one embodiment of the first aspect, the system further comprisesreservoirs for containing the materials, the reservoirs being fluidlycoupled respectively to the microfluidic channels in the print head.

In one embodiment of the first aspect, the print head further comprisesat least two inlets for receiving the materials from the reservoirs,each of the inlets being in fluid communication with respectivemicrofluidic channels and the respective reservoirs.

In one embodiment of the first aspect, the dispensing means comprises apressure control unit.

In one embodiment of the first aspect, the fluidic switches comprisevalves.

In one embodiment of the first aspect, the print head further comprisesa hollow projection configured to extend from the orifice toward thereceiving surface.

In one embodiment of the first aspect, the print head comprises twosecond channels, each of the second channels being adapted to conveyrespective hydrogels, the two second channels intersecting at a secondintersection and joining together at the second intersection to form athird channel which extends to the first intersection point.

In a second aspect, a system for additive manufacturing ofthree-dimensional structures is provided, the system comprising at leastone a print head for receiving and dispensing materials, the materialscomprising a sheath fluid and a hydrogel. In one embodiment, the printhead comprises an orifice for dispensing the materials; microfluidicchannels for receiving and directing the materials to the orifice; andfluidic switches, each fluidic switch corresponding to one of themicrofluidic channels in the print head and configured to allow ordisallow fluid flow in the microfluidic channels in the print head whenactuated. In one embodiment, the system further comprises a receivingsurface for receiving the materials dispensed from the orifice; a fluidremoval feature for removing excess sheath fluid dispensed from theorifice; a positioning unit for positioning the orifice of the printhead in three dimensional space, the positioning unit operably coupledto the print head; and a dispensing means for dispensing the materialsfrom the orifice of the print head.

In one embodiment of the second aspect, the fluid removal featurecomprises a vacuum for drawing the excess sheath fluid away from orthrough the receiving surface and/or from the hydrogel dispensed on thereceiving surface.

In one embodiment of the second aspect, the receiving surface comprisesa porous membrane comprising pores sized to permit passage of the excesssheath fluid there through.

In one embodiment of the second aspect, the vacuum is applied below theporous membrane. In one embodiment of the second aspect, the vacuum isapplied above the receiving surface.

In one embodiment of the second aspect, the vacuum is applied throughone or more vacuum channels provided on the print head, the one or morevacuum channels having an orifice situated near the orifice of the printhead.

In one embodiment of the second aspect, the fluid removal featurecomprises an absorbent material for drawing away from the receivingsurface the excess sheath fluid.

In one embodiment of the second aspect, the system further comprises aprogrammable control processor for controlling the positioning unit andfor controlling dispensing of the materials from the print head onto thereceiving surface.

In one embodiment of the second aspect, the print head further comprisesa hollow projection configured to extend from the orifice toward thereceiving surface.

In one embodiment of the second aspect, the print head comprises one ormore first channels for receiving and directing the sheath fluid and oneor more respective second channels for receiving and directing thehydrogel, the second channels intersecting at a first intersection pointwith the first channels, the second and first channels joining togetherat the first intersection point to form a dispensing channel whichextends to the orifice.

In one embodiment of the second aspect, the print head comprises twosecond channels, each of the second channels being adapted to conveyrespective hydrogels, the two second channels intersecting at a secondintersection and joining together at the second intersection to form athird channel which extends to the first intersection point

In a third aspect, a method of printing a three-dimensional (3D)structure is provided, the method comprising providing a 3D printer, theprinter comprising: a print head comprising an orifice for dispensingmaterials; a receiving surface for receiving a first layer of thematerials dispensed from the orifice of the print head; and apositioning unit operably coupled to the print head, the positioningunit for positioning the print head in three dimensional space. In oneembodiment, the method comprises providing the materials to bedispensed, the materials to be dispensed comprising a sheath fluid andone or more hydrogels; encoding the printer with a 3D structure to beprinted; dispensing from the print head orifice the materials to bedispensed; depositing a first layer of the dispensed materials on thereceiving surface; repeating the depositing step by depositingsubsequent dispensed material on the first and any subsequent layers ofdeposited material, thereby depositing layer upon layer of dispensedmaterials in a geometric arrangement according to the 3D structure; andremoving excess sheath fluid dispensed by the print head orifice at oneor more time point during or between depositing steps.

In one embodiment of the third aspect, the sheath fluid comprises across-linking agent suitable for cross-linking and solidifying thehydrogel upon contact therewith, the contact creating a hydrogel fiber.

In one embodiment of the third aspect, the sheath fluid and the hydrogelare dispensed in a coaxial arrangement, wherein the sheath fluidenvelops the hydrogel.

In one embodiment of the third aspect, the depositing step and theremoving step are carried out continuously, thereby continuouslyremoving the excess sheath fluid as the layers of dispensed materialsare deposited.

In one embodiment of the third aspect, the removing step is carried outintermittently between and/or at the same time as the depositing step,thereby intermittently removing the excess sheath fluid as the layers ofdispensed materials are deposited.

In one embodiment of the third aspect, the one or more hydrogels areadapted for supporting growth and/or proliferation of living cellsdispersed therein.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the invention will become more apparent in the followingdetailed description in which reference is made to the appended drawingswherein:

FIG. 1 is a perspective view of one embodiment of the printing system ofthe present invention.

FIG. 2 is a perspective view of software-designed objects andcorresponding objects printed using one embodiment of the printingsystem of the present invention.

FIG. 3 is a perspective view of one embodiment of the print head of thepresent invention.

FIG. 4 is a cross-section of a valve in the print head of FIG. 3,including deflection of a valve membrane when the valve is actuated.

FIG. 5 is a cross-section of an alternate embodiment of the print headof FIG. 3.

FIG. 6 is a top view of an alternate embodiment of the print head ofFIG. 3.

FIG. 7 is an exploded perspective view of one embodiment of theprint-bed assembly of the present invention.

FIG. 8 is a cross-section of the assembled print-bed of FIG. 9.

FIG. 9 is a cross-section of an alternate embodiment of the print-bed ofFIG. 9.

FIG. 10 is a perspective view of one embodiment of the print head of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending uponthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art, given the context inwhich it is used, “about” will mean up to plus or minus 10% of theenumerated value.

As used herein, the term “hydrogel” refers to a composition comprisingwater and a network or lattice of polymer chains that are hydrophilic.Examples of natural hydrogels include, for example, alginate, agarose,collagen, fibrinogen, gelatin, chitosan, hyaluronic acid based gels orany combination thereof. A variety of synthetic hydrogels are known andcould be used in embodiments of the systems and methods provided herein.For example, in embodiments of the systems and method provided herein,one or more hydrogels form the structural basis for three dimensionalstructures printed. In some embodiments, the hydrogel has the capacityto support growth and/or proliferation of one or more cell types, whichmay be dispersed within the hydrogel or added to the hydrogel after ithas been printed in a three dimensional configuration. In someembodiments, the hydrogel is cross-linkable by a chemical cross-linkingagent. For example, a hydrogel comprising alginate may be cross-linkablein the presence of a divalent cation, a hydrogel comprising fibrinogenmay be cross-linkable in the presence of thrombin, and a hydrogelcomprising collagen or chitosan may be cross-linkable in the presence ofheat or a basic solution. Cross-linking of the hydrogel will increasethe hardness of the hydrogel, in some embodiments allowing formation ofa hydrogel that behaves like a solid.

As used herein, the term “sheath fluid” refers to a liquid that is used,at least in part, to envelope or “sheath” a material to be dispensed,such as, for example, a hydrogel. In some embodiments, the sheath fluidcomprises one or more of an aqueous solvent, for example water orglycerol, and a chemical cross-linking agent, for example materialscomprising divalent cations (e.g. Ca²⁺, Ba²⁺, Sr²⁺, etc.), thrombin, orpH modifying chemicals such as sodium bicarbonate.

As used herein, the term “excess sheath fluid” refers to a portion ofthe sheath fluid that is dispensed from the print head orifice and doesnot form part of a three dimensional structure printed using one or moreembodiments of the systems or methods provided herein. For example, theexcess sheath fluid may be useful in lubricating passage of the hydrogelthrough a dispensing channel in the print head and through the printhead orifice. Once dispensed from the print head orifice the excesssheath fluid may run off of the surface of a layer of dispensed hydrogeland onto a receiving surface, where it may collect or pool.

As used herein, the term “receiving surface” refers to the surface uponwhich a first layer of material dispensed from a print head orifice isdeposited. The receiving surface also receives excess sheath fluid thatis dispensed from the print head orifice and that runs off of one ormore layers of material dispensed from the print head orifice. In someembodiments, the receiving surface is made of a solid material. In someembodiments, the receiving surface is made of a porous material. Forexample, in some embodiments, the porosity of the porous material issufficient to allow passage of the sheath fluid there through. In someembodiments, the receiving surface is substantially planar, therebyproviding a flat surface upon which a first layer of dispensed materialcan be deposited. In some embodiments, the receiving surface has atopography that corresponds to the three dimensional structure to beprinted, thereby facilitating printing of a three dimensional structurehaving a non-flat first layer.

In one aspect, the present invention generally relates to an apparatus,system and method for additive manufacturing of three-dimensional (3D)biological structures.

General Description of the Printing System

In an aspect, the invention provides a system for additive manufacturingof three-dimensional structures (also referred to herein as a “printer”,a “3D printer” or a “printing system” or “the system”). The systemcomprises a microfluidic print head, which is a microfluidic liquidhandling device comprising one or more microfluidic channels forreceiving and directing materials to be dispensed, fluidic switchescorresponding to the microfluidic channels for regulating flow of thematerials to be printed, and a single orifice for dispensing thematerials to be dispensed.

The materials to be dispensed comprise a sheath fluid and at least onehydrogel. In a preferred embodiment, the sheath fluid comprises achemical cross-linking agent suitable for solidifying the hydrogel uponcontact therewith. In a preferred embodiment, the sheath fluid alsoserves as a lubricant for the solidified hydrogel.

The microfluidic channels serve as conduits for directing and combiningthe materials to be dispensed in a controlled manner. The microfluidicchannels are arranged within the print head such that one or more firstchannels for receiving and directing the sheath fluid and a secondchannel for receiving and directing the hydrogel intersect at a firstintersection point and join together to form a dispensing channel whichextends to the orifice of the print head. In one preferred embodiment,the first channels are configured to flank the second channel at thefirst intersection point. In this way, the sheath fluid is directed toflow along either side of the hydrogel in the dispensing channel.

In a preferred embodiment, materials in the dispensing channel aredirected coaxially, the hydrogel being focussed to the center of thedispensing channel and the sheath fluid surrounding the hydrogel fluid,thereby forming a sheath around the hydrogel. In preferred embodimentswhere the sheath fluid also comprises a chemical cross-linking agentsuitable for cross-linking the hydrogel, a solidified hydrogel fiber isformed in the dispensing channel and dispensed from the orifice of theprint head.

In one aspect, the system further comprises a receiving surface forreceiving a first layer of the materials dispensed from the orifice anda positioning unit for positioning the orifice of the print head inthree dimensional space, the positioning unit operably coupled to theprint head. For example, the print head can be coupled to a commerciallyavailable motorized positioning system with three degrees of motion sothat the print head can be positioned above the receiving surface andoriented to direct dispensed material downward towards the receivingsurface.

In one aspect, the system comprises a means for dispensing the materialsfrom the print head orifice and may further comprise and/or be in datacommunication with a programmable control processor for regulatingpositioning of the print head orifice. The programmable controlprocessor may also be used for regulating dispensing of the materials tobe dispensed from the print head orifice.

FIG. 1 shows a schematic perspective view of one embodiment of the 3Dprinting system provided herein.

Referring to FIG. 1, the system comprises a microfluidic print head[100], which comprises a print head orifice [114] and at least one inletfor receiving material to be dispensed from the print head [100]. Thematerial to be dispensed is stored in printed material reservoirs [110]and delivered to the print head through respective first connectingtubes [122], which provide fluid communication between the print headand the printed material reservoirs. In the illustrated embodiment, themeans for dispensing the material to be dispensed from the print headorifice is a pressure control unit [112], which is fluidly coupled tothe printed material reservoirs [110] by respective second connectingtubes [120]. The pressure control unit is a means for providing a forceto dispense the materials to be dispensed. The pressure control unitsupplies pneumatic pressure to the printed material reservoirs [110] viarespective second connecting tubes [120]. The pressure applied to theprinted material reservoirs forces fluid out of the reservoirs and intothe print head via respective first connecting tubes [122]. Alternativemeans for dispensing the material to be dispensed could be used in theillustrated embodiment. For example, a series of electronicallycontrolled syringe pumps could be used to provide force for dispensingthe material to be dispensed from the print head orifice.

Referring to FIG. 1, the microfluidic print head [100] is coupled to a3D motorized stage comprising three arms [102, 103 and 104] forpositioning the print head and the print head orifice [114] in threedimensional space above a print bed [108], which comprises a surface[109] for receiving printed material. In one embodiment, the 3Dmotorized stage (Le., the positioning unit) can be controlled toposition a vertical arm [104], which extends along the z-axis of the 3Dmotorized stage such that the print head orifice is directed downward. Afirst horizontal arm [102], which extends along the x-axis of themotorized stage is secured to an immobile base platform [116]. A secondhorizontal arm [103], which extends along the y-axis of the motorizedstage is moveably coupled to an upper surface of the first horizontalarm [102] such that the longitudinal directions of the first and secondhorizontal arms [102 and 103] are perpendicular to one another. It willbe understood that the terms “vertical” and “horizontal” as used abovewith respect to the arms are meant to describe the manner in which theprint head is moved and do not necessarily limit the physicalorientation of the arms themselves.

In the embodiment illustrated in FIG. 1, the print-bed [108] ispositioned on top of a platform [118], the platform being coupled to anupper surface of the second horizontal arm [103]. In the embodiment, the3D motorized stage arms [102,103 and 104] are driven by threecorresponding motors [105, 106 and 107], respectively, and controlled bya programmable control processor, such as a computer (not shown). In apreferred embodiment, the print head [100] and print-bed [108] arecollectively moveable along all three primary axes of a Cartesiancoordinate system by the 3D motorized stage and movement of the stage isdefined using computer software.

It will be understood that the invention is not limited to only thedescribed positioning system and that other positioning systems areknown in the art.

In the embodiment illustrated in FIG. 1, as material is dispensed fromthe print head orifice [114], the positioning unit is moved in a patterncontrolled by software, thereby creating a first layer of the dispensedmaterial on the receiving surface [109]. Additional layers of dispensedmaterial are stacked on top of one another such that the final 3Dgeometry of the dispensed layers of material is generally a replica ofthe 3D geometry design provided by the software. The 3D design may becreated using typical 3D CAD (computer aided design) software orgenerated from digital images, as known in the art. Further, if thesoftware generated geometry contains information on specific materialsto be used, it is possible, according to one embodiment of theinvention, to assign a specific material type to different geometricallocations. For example, FIG. 2 shows three 3D structures printed usingone embodiment of the system provided herein: a cube [128], a hollowcylinder [129] and a hollow coaxial cylinder [130]. Software was used togenerate cube, hollow cylinder and hollow coaxial cylinder designs([125], [126] and [127], respectively), each design comprising twodifferent types of materials (dyed alginate), which were dyed differentcolors to provide visual clarity of the materials used to generate theprinted cube and hollow cylinder.

Any software, application or module referred to herein may beimplemented using computer readable/executable instructions that may bestored or otherwise held by such computer readable media.

Print Head

FIG. 3 shows a schematic perspective view of one embodiment of amicrofluidic print head [100] for use in the system provided.

Referring to FIG. 3, the illustrated embodiment depicts a microfluidicprint head [100] comprising microfluidic channels for carrying variousfluids. In the illustrated embodiment, the microfluidic channels have acylindrical shape. However, channel shapes other than cylindrical couldalso be used in the print head provided herein. Channel [200] is aconduit for a cross-linking agent, channel [202] is a conduit for water.In the illustrated embodiment, the cross-linking agent and water,separately or together serve as the “sheath fluid”. Channel [204] is aconduit for a first hydrogel composition (referred to as “hydrogel A”),and channel [206] is a conduit for a second hydrogel composition(referred to as “hydrogel B”). In a preferred embodiment, one or moreliving cell types are compatible with and optionally dispersed withinhydrogels A and/or B. In the illustrated embodiment, each microfluidicchannel comprises a fluid inlet [208 a , 208 b , 208 c , 208 d ], whichallows fluid contained in the connecting tubes [122] to pass into therespective channels of the print head [100]. Downstream of the fluidinlets [208 a , 208 b , 208 c , 208 d ] are valves [210, 212, 214, 216]corresponding to each channel. In the illustrated embodiment, the valvesserve as “fluidic switches”, which can be actuated to allow and disallowflow of fluid through a channel, each valve having a corresponding inlet[218, 218 a , 218 b , 218 c , 218 d ], which facilitates actuation andde-actuation of the valve. In one embodiment, the valves [210, 212, 214,216] can be electronically actuated. In another embodiment, the valves[210, 212, 214, 216] can be actuated by a change in applied pressure,for example, by way of solenoid pistons. Electronic or pressureactuation of different valves facilitates rapid change of the materialdispensed, thereby allowing the materials dispensed to be composed of acontrolled sequence of different materials.

Referring further to FIG. 3, in the illustrated embodiment, thecrosslinking agent channels [200] and water channels [202] intersect atintersection points [203], such as in a “y-shaped” configuration,joining together to form channels referred to herein as “sheath flowchannels” [224] immediately downstream of the crosslinking agent andwater channels [200, 202]. The hydrogel A and hydrogel B channels [204,206] intersect at an intersection point [207], such as in a “y-shaped”configuration, joining together to form a channel referred to herein asa “focussing channel” [226] immediately downstream of the two hydrogelchannels. The sheath flow channels [224] and the focussing channel [226]intersect at an intersection point [228] in a three-prongedconfiguration, for the described embodiment, wherein the focussingchannel [226] is flanked by the sheath flow channels [224], joiningtogether to form a channel referred to herein as a dispensing channel[220]. The dispensing channel [220] terminates in the dispensing orifice[222]. In a preferred embodiment illustrated in FIG. 1, the dispensingchannel projects from the print head [100] terminating in the dispensingorifice [114].

Referring further to FIG. 3, in the illustrated embodiment, the sheathflow channels [224] and the dispensing channel [220] have largerdiameters than the focussing channel [226]. When hydraulic pressure isapplied to the sheath flow [224] and focussing channels [226], liquid inthe focussing channel [226] is compressed laterally and “focussed” intoa narrow stream along the central axis of the focussing channel [226].Upon intersection with the focussing channel [226] at the intersectionpoint [228], fluid from the larger diameter sheath flow channels [224]surrounds and envelopes the narrower focussed stream of hydrogeldispensed from the focussing channel [226].

In a preferred embodiment, liquid in the sheath flow channels [224]comprises a chemical cross-linking agent and liquid in the focussingchannel [226] comprises one or more chemically cross-linkable hydrogelscomprising one or more living cell types. When the one or morechemically cross-linkable hydrogels are focussed into a narrow stream inthe focussing channel [226] and then enveloped by the cross-linkingagent in the dispensing channel [220], at least the exterior surface ofthe one or more chemically cross-linkable hydrogels is solidified in thedispensing channel [220], thereby creating a cross-linked or “solid”hydrogel fiber. The hydrogel fiber is then dispensed from the dispensingorifice [222] onto the receiving surface in a controlled manner,building a 3D structure, layer by layer.

In a particularly preferred embodiment, the sheath fluid surrounding thehydrogel fiber may also act to lubricate passage of the hydrogel fiberthrough the dispensing channel [220] until it is dispensed from theprint head orifice [222].

In an embodiment, the sheath fluid comprises a chemical cross-linkingagent, water or a combination thereof. In embodiments where the sheathfluid lacks a chemical cross-linking agent the hydrogel will not besolidified and would be dispensed as a liquid. In order to adjust thecomposition of the sheath fluid and start and/or stop solidification ofthe hydrogel, a crosslinking agent channel valve [210] and water channelvalve [212] may be actuated. It is contemplated that dispensing a liquidrather than a solid hydrogel, or dispensing sheath fluid alone, may bedesirable in order to construct some aspects of various threedimensional objects.

In an embodiment, the print head [100] may be configured to receive anddispense only one hydrogel material. In one embodiment, the print headmay be configured to receive and dispense two or more hydrogelmaterials. For example, in an embodiment where the print head [100] isconfigured to receive two hydrogel materials, each, for example,comprising a different cell type, the system provided herein can beprogramed to dispense a heterogeneous cellular structure, wherein firstand second cell types can be laid down in controlled patterns within andamong layers, alone and/or in combination with one another. Boundariesbetween the two materials are controlled, e.g., by software, and theprogrammable control processor is used to instruct fluidic switched(e.g., one or more of valves [210], [212], [214], [216]) to change theflow of material within one or more microfluidic channels, therebychanging the content of the material being dispensed from the print headorifice. The number of hydrogel materials that can be received by anddispensed from the print head provided herein is limited only by thesize of the print head that the user deems practical.

Referring to FIG. 4, in one embodiment, the fluidic switch is a valvecomprising a membrane [332] disposed over a bowl-shaped feature [318]formed in a microfluidic channel [308]. Upon application of pneumaticpressure (represented by arrows in FIG. 4) to the exposed surface of thevalve membrane [332], the valve membrane [332] will be deflected intothe bowl shaped feature [318], thereby blocking passage of fluid throughmicrofluidic channel [308]. In one preferred embodiment, the thicknessof the valve membrane [332] is about 150 μm. In embodiments where thevalve membrane thickness is increased, a skilled person would understandthat the applied pneumatic valve actuation pressure must be increasedaccordingly. Similarly, a valve membrane formed of less resilientmaterial will require a higher actuation pressure. A skilled personwould understand how to adjust the actuation pressure to suit thespecific material of the valve membrane.

In one embodiment, the print head comprises alternative fluidic switchesfor regulating materials to be dispensed from the print head orifice.For example, rather than using valves, a mechanism for engaging ordisengaging the pressure applied to each channel could be used toregulate material flow in the microfluidic channels.

In one embodiment, the print head further comprises an extension tipcomprising an orifice for dispensing materials from the print head. Suchan extension tip facilitates precision dispensing of materials anddeposition thereof in confined areas such as, for example, a well in amulti-well plate (e.g., a standard microtitre plate, microwell plate ormicroplate having 6, 24, 96 etc. wells) or a petri dish. Referring tothe embodiment illustrated in FIG. 5, a portion [500] of the dispensingchannel [220] nearest to the dispensing orifice [222] has a largerdiameter than the upstream portion of the dispensing channel [220]. Theextension tip [502] comprises a tube (e.g., made of plastic, glass ormetal) having an exterior configured to fit into the large-diameterportion [500] of the dispensing channel and an inner surface (defining ahollow space in the tube) configured to align with the dispensingchannel [220]. The extension tip [502] can be inserted into thelarge-diameter portion [500] of the dispensing channel, thereby,extending the length of the dispensing channel [220], which facilitatesdeposition of material dispensed from an orifice [503] in the extensiontip [502] into confined spaces, such as a well plate insert [504] orpetri dish (not shown).

Referring to the embodiment illustrated in FIG. 1, the extension tip[130] is a projection extending from the print head [100], the extensiontip [130] terminating in the print head orifice [114]. In thisembodiment, the extension tip [130] is integral with the print head.

In one embodiment, two or more hydrogel materials can be arrangedcoaxially in a hydrogel fiber dispensed from the system provided herein.Referring to FIG. 6, in the illustrated embodiment, the print head [100]comprises microfluidic channels arranged to produce a coaxial hydrogelfiber comprising a hydrogel core material and hydrogel shell material.In the illustrated embodiment, the shell material, carried in channels[508], is a rapidly gelling hydrogel, such as alginate, and the corematerial, carried in channel [506], is a different hydrogel chosen bythe user (e.g. collagen or fibrinogen). Channels [508] and channel [506]intersect at a hydrogel focussing intersection point [510], for examplein a “y-shaped” configuration (similar to intersection [528] shown inFIG. 3) joining together to form a focussing channel [226] downstream ofchannels [506] and [508]. At the hydrogel focussing intersection [510],the shell material focusses the core material coaxially such that theshell material forms a sheath around the core material. In preferredembodiments, channels [508] and [226] have a larger diameter thanchannel [506] to facilitate coaxial focussing of the core and shellmaterials. In a preferred embodiment, the purpose of the shell materialis to provide the core material with physical structural support so thatit may be formed into a 3D geometry. The core may be solidified afterthe material is deposited, the precise method of solidification beingspecific to different core materials. For example, the core may comprisea material that solidifies very slowly. In another embodiment, the coreand shell materials comprise the same materials. In yet anotherembodiment, the shell material comprises a hydrogel that rapidlysolidifies and the core material comprises a material that will not gel,thereby facilitating generation of a hollow fiber.

In one embodiment, the print head [100] depicted in FIG. 6 could furthercomprise additional core material channels, each with a correspondingfluidic switch, for example a valve, for regulating flow of the materialtherein. The fluidic switch facilitates rapid and frequent adjustmentsto the composition of the core material in the fiber being dispensed,for example, by commands provided by the programmable control processor.

In one embodiment, several print heads could be arranged, for example inparallel, to allow simultaneous printing of multiple structures. Thiswould increase throughput production.

In some embodiments the print head is disposable. Use of disposableprint heads can reduce the likelihood of contamination of materials usedin different print jobs.

The print head can be fabricated, for example, using known microfluidicsmolding techniques (e.g., casting, imprinting or injection molding) andone or more moldable polymers, for example, polydimethylsiloxane (PDMS).Alternatively, commercially available 3D printing technology could beused to fabricate the print head.

Fluidic Removal Feature

In an aspect, the invention provides a system for additive manufacturingof three-dimensional structures that comprises a feature for removingexcess sheath fluid from the receiving surface where a first layer ofmaterial dispensed from the orifice of the print head is deposited andoptionally from a surface of dispensed hydrogel. During printing, it ispossible that excess sheath fluid will collect or “pool” on thereceiving surface or on a surface of dispensed hydrogel. Such poolingcan interfere with deposition of hydrogel dispensed from the print headorifice onto the receiving surface and/or onto one or more layers ofdispensed hydrogel. For example, pooled sheath fluid may cause adispensed hydrogel fiber to slip from its intended position in the 3Dstructure being printed. Therefore, in embodiments of the system,removal of excess sheath fluid from the receiving surface and optionallyfrom a surface of the dispensed hydrogel by way of a fluidic removalfeature may improve additive manufacturing of three-dimensionalstructures.

Excess sheath fluid may be removed from the receiving surface or from asurface of one or more layers of dispensed hydrogel by drawing the fluidoff of those surfaces, by allowing or facilitating evaporation of thesheath fluid from those surfaces or, in embodiments where the receivingsurface is porous, excess sheath fluid may be removed by drawing itthrough the porous surface.

In a preferred embodiment, the receiving surface comprises a porousmaterial, the pores being sized to facilitate passage of sheath fluidthere through and sized to support one or more layers of hydrogeldeposited thereon.

Referring to FIGS. 7 and 8, in the illustrated embodiments, a print bed[108] comprises a porous membrane [400], which serves as the surface forreceiving a first layer of dispensed material (i.e., the receivingsurface). The porous membrane [400] is held in place in the print bed[108] between a box piece [408] and a lid piece [402]. The box piece[408] is a container, which can be any shape suitable for receiving andcontaining liquid (e.g., square, round). The space inside of the boxpiece [408] is referred to as a chamber [404]. The box piece [408] hasan upper surface [409] comprising a recessed lip [412] extending theperimeter of the upper surface [409] of the box piece [408]. The uppersurface [409] comprises an aperture defined by one or more walls [410],the aperture being surrounded by the recessed lip [412] and extendinginto the box piece [408].

Referring further to the embodiments illustrated in FIGS. 7 and 8, thelid piece [402] comprises an upper surface [403] having an aperture[416] that extends therethrough and sidewalls [418] configured to fitaround the recessed lip [412] of the box piece [408], therebyfacilitating placement of the lid piece [402] on the upper surface [409]of the box piece [408]. When the lid piece [402] is placed on the boxpiece [408] apertures in the box and the lid piece [416] align. Inoperation, the porous membrane [400] is placed on the upper surface[409] of the box piece [408] such that it extends over the aperture inthe upper surface [409] of the box piece [408], the lid piece [402] isthen placed on top of the box piece and pressed downward. The downwardpressure of the lid piece [402] stretches the porous membrane [400] overthe aperture in the upper surface [409] of the box piece [408], therebyretaining the porous membrane [400] between the box piece [408] and thelid piece [402]. In preferred embodiments, the lid piece [402] and boxpiece [408] fit together snuggly, thereby providing a connection thatwill remain secure during operation of the system provided herein.

Referring further to the embodiments illustrated in FIGS. 7 and 8, thebox piece [408] comprises a solid base [414] and at least one outletduct [406] for directing fluid away from the chamber [404], and a vacuumsource (not shown) in fluid communication with the outlet duct [406] ofthe chamber [404]. The porous membrane [400] comprises pores sized tofacilitate passage of sheath fluid. The vacuum source (not shown)coupled to the outlet duct [406] may be actuated to draw the excesssheath fluid collected on the porous membrane [400] through the porousmembrane [400] into the chamber [404] and from the chamber [404] throughthe outlet [406], leaving the hydrogel fiber in its dispensedconfiguration on top of the porous membrane [400].

In a preferred embodiment, a feature for removing excess sheath fluidfrom the receiving surface and optionally from a surface of dispensedhydrogel can be included in a system configured to dispense materialsinto a multiwall plate or petri dish. For example, referring to FIG. 9,in the illustrated embodiment, a commercially available well-plateinsert [504], is placed on top of the box piece [408]. Some well-plateinserts [504] have a basket shape with a base made out of a porousmembrane material [512]. In the illustrated embodiment, a gasket [514]is placed between the well-plate insert [512] and the box piece [408] toimprove sealing between the two pieces [504 and 408]. In suchembodiments, the porous membrane [512] of the well-plate inset [504]would serve as the “receiving surface” and any excess sheath fluid couldbe removed therefrom using a vacuum coupled to the outlet duct [406], asdescribed above, or using one of the other fluidic removal featuresdescribed below.

In one embodiment (not shown), the receiving surface on the print bedcomprises or is placed adjacent to an absorptive material, whichfacilitates absorption of excess sheath fluid from the receivingsurface. For example, a well-plate insert having a base made out of aporous membrane material (for example, as shown in FIG. 9), or any otherporous membrane substrate, could be placed on top of or adjacent to anabsorptive material, such as, for example, a sponge. The absorptivematerial would act to draw away from the receiving surface excess sheathfluid. In embodiments where the absorbent material is disposed below aporous receiving surface, excess sheath fluid on the receiving surfacewould be drawn through the porous receiving surface and into theabsorptive material, thereby preventing pooling of excess sheath fluidon the receiving surface. In embodiments where the absorbent material isdisposed immediately beside or on top of a portion of the receivingsurface (e.g., on the periphery of the receiving surface so as not tointerfere with deposition of dispensed material) excess sheath fluidwould be drawn off of the receiving surface and into the absorbentmaterial.

In one embodiment (not shown), rather than using one of the print bedsdescribed above, one or more tubes may be provided in an area near thereceiving surface and near the print head orifice. The one or more tubesmay be fluidly coupled to a vacuum source (not shown), which can providesuction for removing excess sheath fluid from the receiving surface andoptionally from a surface of dispensed hydrogel. In such embodiments, asolid or porous receiving surface may also be used.

In one embodiment, illustrated in FIG. 10, the print head is configuredto further comprise one or more vacuum channels [700 a , 700 b ], theone or more vacuum channels each having an orifice [702 a , 702 b ]situated near the print head orifice [222]. The one or more vacuumchannels [700 a , 700 b ] each have an inlet [704 a , 704 b ] configuredto facilitate fluid communication with one or more vacuums (not shown).When the print head [100] is in fluid communication with a vacuum, theone or more vacuum channels [702 a , 702 b ] direct negative pressure toan area of the receiving surface where materials are being dispensed orhave been dispensed from the print head orifice [222] and/or to aportion of the surface area of the dispensed hydrogel, thereby drawingup excess sheath fluid from the receiving surface and optionally from asurface of the dispensed hydrogel, thereby eliminating pooling of sheathfluid on the receiving surface and/or the dispensed hydrogel.

In one embodiment, the one or more vacuum tubes are provided, at leastin part, in one or more extensions projecting from the print head, theextensions projecting in the same general direction as the extensioncomprising the print head orifice and dispensing channel (see, forexample, FIG. 10). In such embodiments, the one or more extensionscomprising vacuum tubes do not extend further than the extensioncomprising the print head orifice and dispensing channel so as not tointerfere with dispensed and deposited hydrogel.

It is contemplated that in some embodiments, the fluid removal featuremay be a feature of the sheath fluid composition itself. For example,the sheath fluid composition may be designed to evaporate after it isdispensed from the print head orifice, thereby eliminating pooling ofexcess sheath fluid on the receiving surface or on surfaces of dispensedhydrogel. For example, the sheath fluid may have a boiling point thatresults in evaporation after being dispensed, while remaining in aliquid state prior to being dispensed.

Method of Printing a Three Dimensional Structure

In an aspect, a method of printing a three-dimensional (3D) structure isprovided.

The method first comprises providing a design for a 3D structure to beprinted. The design may be created using commercially available CADsoftware. In one embodiment, the design comprises information regardingspecific materials (e.g., for heterogeneous structures comprisingmultiple materials) to be assigned to specific geometrical locations inthe design.

The method comprises the use of a 3D printer, the printer comprising: aprint head, a receiving surface for receiving material dispensed by theprint head; and a positioning unit operably coupled to the receivingsurface, the positioning unit for positioning the print head at alocation in three dimensional space above the receiving surface. Forexample, various embodiments of the printing system provided herein maybe used in the method of printing a 3D structure.

The method comprises providing at least two materials to be dispensed bythe print head, such as a sheath fluid and a hydrogel fluid. Inpreferred embodiments, one or more cell types are compatible with, andoptionally dispensed within, the hydrogel. In a preferred embodiment,the sheath fluid serves as a lubricating agent for lubricating movementof the hydrogel within and from the print head. In a preferredembodiment, the sheath fluid comprises a cross-linking agent forsolidifying at least a portion of the hydrogel before or while it isdispensed from the print head.

The method comprises communicating the design to the 3D printer.Communication can be achieved, for example, by a programmable controlprocessor.

The method comprises controlling relative positioning of the print headand the receiving surface in three dimensional space and simultaneouslydispensing from the print head the sheath fluid and the hydrogel, aloneor in combination. In preferred embodiments, the materials dispensedfrom the print ahead are dispensed coaxially, such that the sheath fluidenvelopes the hydrogel. Such coaxial arrangement allows thecross-linking agent to solidify the hydrogel, thereby resulting in asolid hydrogel fiber, which is dispensed from the printer head.

The method comprises depositing a first layer of the dispensed materialson the receiving surface, the first layer comprising an arrangement ofthe material specified by the design and iteratively repeating thedepositing step, depositing subsequent material onto the first andsubsequent layers of material, thereby depositing layer upon layer ofdispensed materials in a geometric arrangement specified by the designto produce the cell-laden 3D structure.

In preferred embodiments, a plurality of materials, for example multiplehydrogels, at least some of which comprise one or more cell types, aredeposited in a controlled sequence, thereby allowing a controlledarrangement of hydrogels and cell types to be deposited in a geometricarrangement specified by the design.

In preferred embodiments, the method comprises removing excess sheathfluid from the receiving surface and optionally from the surface of thedispensed hydrogel. For example, the step of removing the excess sheathfluid can be done continuously throughout the printing process, therebyremoving excess fluid that may otherwise interfere with layering thedispensed materials in the geometric arrangement provided by the design.Alternatively, the step of removing excess sheath fluid may be doneintermittently throughout the printing process in sequence with orsimultaneously with one or more depositing steps. In some embodiments,removal of excess sheath fluid is achieved by drawing the fluid off ofthe receiving surface and optionally off of a surface of the dispensedhydrogel. In another embodiment removal of excess sheath fluid isachieved by drawing excess fluid through the receiving surface, thereceiving surface comprising pores sized to allow passage of the sheathfluid. In another embodiment removal of excess sheath fluid is achievedby providing a sheath fluid that evaporates after being dispensed fromthe print head orifice.

Exemplary Uses of Embodiments of the System and Method of PrintingCell-Laden Three Dimensional Structures

In some embodiments, structures generated using the system and methodprovided herein can be useful in the field of drug discovery, where, forexample, determining cellular responses to various chemical compoundsand compositions are of interest. Use of 3D cell cultures fabricatedusing embodiments of the systems and methods provided herein may provideexperimental conditions that more closely resemble in vivo cellular andtissue conditions relative to 2D cell cultures. 3D arrangement of thecells may more closely mimic in vivo cell-cell interactions andresponses to external stimuli and the heterogeneous nature of the 3Dstructures that can be generated using the apparatus and methodsprovided permit study of tissues and potentially organs. It iscontemplated that 3D cell-laden structures fabricated using embodimentsof the systems and methods provided herein may provide a similar benefitto the cosmetics industry by offering an alternative means to testingcosmetic products.

In some embodiments, various embodiments of the system and methodprovided herein are compatible with standard well-plate technology.Well-plates or well-plate inserts may be used with or as part of theprint bed in the methods and systems provided herein. Variousembodiments of the system and method provided herein are thus compatiblewith instruments and practices that utilize well-plates, allowing themto be readily integrated into existing process streams.

In some embodiments, the microfluidic channels within the print head arecompatible with other microfluidic modules. For example, knownmicrofluidic modules may be included in the print head of the systemsprovided herein upstream of the print head orifice. Such modules mayinclude, for example, cell counting, cell sorting, cell analyzing,and/or concentration gradient generating modules.

In some embodiments, throughput of 3D printing may be increased byadding to the system additional print heads in parallel. Each print headcomprising all of the elements required to print a multi-materialstructure, thus allowing several 3D structures to be printedsimultaneously by including additional print heads in the system.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the purpose and scope ofthe invention as outlined in the claims appended hereto. Any examplesprovided herein are included solely for the purpose of illustrating theinvention and are not intended to limit the invention in any way. Anydrawings provided herein are solely for the purpose of illustratingvarious aspects of the invention and are not intended to be drawn toscale or to limit the invention in any way. The disclosures of all priorart recited herein are incorporated herein by reference as if set forthin their entirety.

REFERENCES

The following references are provided as examples of the known artrelating to the present invention. The following listing is not intendedto comprise a comprehensive list of all relevant art. The entirecontents of all references listed in the present specification,including the following documents, are incorporated herein by referenceas if set forth in their entirety.

1. Su-Jung Shin, Ji-Young Park, Jin-Young Lee, Ho Park, Yong-Doo Park,Kyu-Back Lee, Chang-Mo Whang, and Sang-Hoon Lee, ““On the fly”continuous generation of alginate fibers using a microfluidic device”,Langmuir, Vol. 23, 2007, pp. 9104-9108.

-   -   2. Saif Khalil, and Wei Sun, “Bioprinting endothelial cells with        alginate for 3D tissue constructs”, Journal of Biomechanical        Engineering, Vol. 131, 2009, pp. 111002-1-111002-8.    -   3. Min Hu, Rensheng Deng, Karl M. Schumacher, Motoichi Kurisawa,        Hongye Ye, Kristy Purnamawati, and Jackie Y. Ying, “Hydrodynamic        spinning of hydrogel fibers”, Biomaterials, Vol. 31, 2010, pp.        863-869.    -   4. Byung Kim, Intae Kim, Wooseok Choi, Sun Won Kim, JooSung Kim,        and Geunbae Lim, “Fabrication of cell-encapsulated alginate        microfiber scaffold using microfluidic channel”, Journal of        Manufacturing Science and Engineering, Vol. 130, 2008, pp.        021016-1-021016-6.    -   5. Edward Kang, Su-Jung Shin, Kwang Ho Lee, and Sang-Hoon Lee,        “Novel PDMS cylindrical channels that generate coaxial flow, and        application to fabrication of microfibers and particles”, Lab on        a Chip, Vol. 10, 2010, pp. 1856-1861.    -   6. Hiroaki Onoe, Riho Gojo, Yukiko Tsuda, Daisuke Kiriyaand, and        Shoji Takeuchi, “Core-shell gel wires for the construction of        large area heterogeneous structures with biomaterials”, IEEE        MEMS Conference, 2010, pp. 248-251.    -   7 Setareh Ghorbanian (2010), Microfluidic probe for direct write        of soft cell scaffolds, M.Eng. Thesis. McGill University:        Canada.    -   8. Edward Kang, Gi Seok Jeong, Yoon Young Choi, Kwang Ho Lee,        Ali Khademhosseini, and Sang-Hoon Lee, “Digitally tunable        physicochemical coding of material composition and topography in        continuous microfibers”, Nature Materials, Vol. 10, 2011, pp.        877-883.    -   9. EP 2489779 A1    -   10. US 2006/0105011 A1    -   11. US 2011/0136162 A1    -   12. US 2012/0089238 A1    -   13. WO 2012009363 A1

We claim:
 1. A system for additive manufacturing of three-dimensionalstructures, the system comprising: at least one print head for receivingand dispensing materials, the materials comprising a sheath fluid and ahydrogel, the print head comprising: an orifice for dispensing thematerials; microfluidic channels comprising one or more first channelsfor receiving and directing the sheath fluid and one or more respectivesecond channels for receiving and directing the hydrogel, the secondchannels intersecting at a first intersection point with the firstchannels, the second and first channels joining together at the firstintersection point to form a dispensing channel which extends to theorifice; and fluidic switches, each fluidic switch corresponding to oneof the microfluidic channels in the print head and configured to allowor disallow fluid flow in the microfluidic channels of the print headwhen actuated; a receiving surface for receiving a first layer of thematerials dispensed from the orifice; a positioning unit for positioningthe orifice of the print head in three dimensional space, thepositioning unit operably coupled to the print head; and a dispensingmeans for dispensing the materials from the orifice of the print head.2. The system of claim 1, further comprising a programmable controlprocessor for controlling the positioning unit and for controllingdispensing of the materials from the print head onto the receivingsurface.
 3. The system of claim 1 or 2, wherein the one or more firstchannels comprise at least two channels, the one or more first channelsbeing configured to flank respective second channels at the firstintersection point.
 4. The system of any one of claims 1 to 3, whereinthe sheath fluid comprises a cross-linking agent for solidifying thehydrogel upon contact therewith at the intersection point and/or in thedispensing channel.
 5. The system any one of claims 1 to 4, wherein eachsecond channel has a diameter less than that of the first channels andthe dispensing channel, whereby flow from the first channels forms acoaxial sheath around the hydrogel in the dispensing channel.
 6. Thesystem of any one of claims 1 to 5, wherein the hydrogel comprisesliving cells.
 7. The system of any one of claims 1 to 5, furthercomprising a fluid removal feature for removing excess sheath fluid fromdispensed from the print head.
 8. The system of claim 7, wherein thereceiving surface comprises a porous membrane comprising pores sized topermit passage of the excess sheath fluid there through.
 9. The systemof claim 8, wherein the fluid removal feature comprises absorbentmaterial or a vacuum for drawing the excess sheath fluid away from thereceiving surface.
 10. The system of claim 9, wherein the absorbentmaterial or vacuum is applied below a porous membrane.
 11. The system ofclaim 9, wherein the vacuum is applied above the receiving surface. 12.The system of claim 11, wherein the vacuum is applied through one ormore vacuum channels provided on the print head, the one or more vacuumchannels having an orifice situated near the orifice of the print head.13. The system of any one of claims 1 to 12, further comprisingreservoirs for containing the materials, the reservoirs being fluidlycoupled respectively to the microfluidic channels in the print head. 14.The system of claim 13, wherein the print head further comprises atleast two inlets for receiving the materials from the reservoirs, eachof the inlets being in fluid communication with respective microfluidicchannels and the respective reservoirs.
 15. The system of any one ofclaims 1 to 14, wherein the dispensing means comprises a pressurecontrol unit.
 16. The system of any one of claims 1 to 15, wherein thefluidic switches comprise valves.
 17. The system of any one of claims 1to 16, wherein the print head further comprises a hollow projectionconfigured to extend from the orifice toward the receiving surface. 18.The system of any one of claims 1 to 17, wherein the print headcomprises two second channels, each of the second channels being adaptedto convey respective hydrogels, the two second channels intersecting ata second intersection and joining together at the second intersection toform a third channel which extends to the first intersection point. 19.A system for additive manufacturing of three-dimensional structures, thesystem comprising: at least one print head for receiving and dispensingmaterials, the materials comprising a sheath fluid and a hydrogel, theprint head comprising: an orifice for dispensing the materials;microfluidic channels for receiving and directing the materials to theorifice; and fluidic switches, each fluidic switch corresponding to oneof the microfluidic channels in the print head and configured to allowor disallow fluid flow in the microfluidic channels in the print headwhen actuated; a receiving surface for receiving the materials dispensedfrom the orifice; a fluid removal feature for removing excess sheathfluid dispensed from the orifice; a positioning unit for positioning theorifice of the print head in three dimensional space, the positioningunit operably coupled to the print head; and a dispensing means fordispensing the materials from the orifice of the print head.
 20. Thesystem of claim 19, wherein the fluid removal feature comprises a vacuumfor drawing the excess sheath fluid away from or through the receivingsurface and/or from the hydrogel dispensed on the receiving surface. 21.The system claim 20, wherein the receiving surface comprises a porousmembrane comprising pores sized to permit passage of the excess sheathfluid there through.
 22. The system of claim 21, wherein the vacuum isapplied below the porous membrane.
 23. The system of claim 20, whereinthe vacuum is applied above the receiving surface.
 24. The system ofclaim 23, wherein the vacuum is applied through one or more vacuumchannels provided on the print head, the one or more vacuum channelshaving an orifice situated near the orifice of the print head.
 25. Thesystem of claim 19, wherein the fluid removal feature comprises anabsorbent material for drawing away from the receiving surface theexcess sheath fluid.
 26. The system of any one of claims 21 to 25,further comprising a programmable control processor for controlling thepositioning unit and for controlling dispensing of the materials fromthe print head onto the receiving surface.
 27. The system of any one ofclaims 21 to 26, wherein the print head further comprises a hollowprojection configured to extend from the orifice toward the receivingsurface.
 28. The system of any one of claims 21 to 27, wherein the printhead comprises one or more first channels for receiving and directingthe sheath fluid and one or more respective second channels forreceiving and directing the hydrogel, the second channels intersectingat a first intersection point with the first channels, the second andfirst channels joining together at the first intersection point to forma dispensing channel which extends to the orifice.
 29. The system ofclaim 28, wherein the print head comprises two second channels, each ofthe second channels being adapted to convey respective hydrogels, thetwo second channels intersecting at a second intersection and joiningtogether at the second intersection to form a third channel whichextends to the first intersection point
 30. A method of printing athree-dimensional (3D) structure, the method comprising: providing a 3Dprinter, the printer comprising: at least one print head comprising anorifice for dispensing materials; a receiving surface for receiving afirst layer of the materials dispensed from the orifice of the printhead; and a positioning unit operably coupled to the print head, thepositioning unit for positioning the print head in three dimensionalspace; providing the materials to be dispensed, the materials to bedispensed comprising a sheath fluid and one or more hydrogels; encodingthe printer with a 3D structure to be printed; dispensing from the printhead orifice the materials to be dispensed; depositing a first layer ofthe dispensed materials on the receiving surface; repeating thedepositing step by depositing subsequent dispensed material on the firstand any subsequent layers of deposited material, thereby depositinglayer upon layer of dispensed materials in a geometric arrangementaccording to the 3D structure; and removing excess sheath fluiddispensed by the print head orifice at one or more time point during orbetween depositing steps.
 31. The method of claim 30, wherein the sheathfluid comprises a cross-linking agent suitable for cross-linking andsolidifying the hydrogel upon contact therewith, the contact creating ahydrogel fiber.
 32. The method of claim 31, wherein the sheath fluid andthe hydrogel are dispensed in a coaxial arrangement, wherein the sheathfluid envelops the hydrogel.
 33. The method of any one of claims 30 to32, wherein the depositing step and the removing step are carried outcontinuously, thereby continuously removing the excess sheath fluid asthe layers of dispensed materials are deposited.
 34. The method of anyone of claims 30 to 32, wherein the removing step is carried outintermittently between and/or at the same time as the depositing step,thereby intermittently removing the excess sheath fluid as the layers ofdispensed materials are deposited.
 35. The method of any one of claims30 to 34, wherein the one or more hydrogels are adapted for supportinggrowth and/or proliferation of living cells dispersed therein.